LncRNA-Braveheart促进骨髓间充质干细胞在体外向心肌样细胞分化

发布时间：2018-03-06 01:00

本文选题：心肌梗塞　切入点：骨髓　出处：《中国组织工程研究》2017年29期 　论文类型：期刊论文

【摘要】：背景:课题组前期研究发现骨髓间充质干细胞移植能够改善大鼠心肌梗死后心功能,但整体效果并不太理想,骨髓间充质干细胞在体内局部梗死微环境中的分化效率低下,其向心肌细胞分化的能力极其有限。目的:采用lncRNA-Bvht体外转染骨髓间充质干细胞,观察其对骨髓间充质干细胞向心肌样细胞分化的影响。方法:构建含lncRNA-Bvht的病毒载体p LVX-IRES-Zs Green1-lncRNA-Bvht,对骨髓间充质干细胞进行lncRNA-Bvht转染并检测转染效率。分离培养C57BL/6小鼠的骨髓间充质干细胞,并将第3代细胞分为3组:骨髓间充质干细胞组、空载体组和lncRNA-Bvht组。各组细胞培养48 h后用5-氮杂胞苷进行诱导分化24 h再进行正常培养2周,荧光显微镜观察骨髓间充质干细胞向心肌细胞分化情况,免疫荧光染色、Western blot、qRT-PCR检测心肌分化标记物肌钙蛋白T和肌节蛋白的表达。结果与结论:①诱导分化后细胞形态发生改变,细胞之间形成连接,排列方向趋于一致;②免疫荧光检测结果显示lncRNA-Bvht组肌钙蛋白T和肌节蛋白表达呈现强阳性;肌钙蛋白T阳性细胞的比例显著增加;③qRT-PCR和Western blot检测结果显示lncRNA-Bvht组肌钙蛋白T和肌节蛋白的表达较空载体组和骨髓间充质干细胞组均明显升高(P0.01);④结果提示,lncRNA-Bvht转染能够有效促进骨髓间充质干细胞在体外向心肌样细胞发生分化。
[Abstract]:Background: our previous study found that bone marrow mesenchymal stem cell transplantation can improve cardiac function after myocardial infarction in rats, but the overall effect is not very good, the differentiation efficiency of bone marrow mesenchymal stem cells in the microenvironment of local infarction in vivo is low. Its ability to differentiate into cardiomyocytes is extremely limited. Objective: to transfect bone marrow mesenchymal stem cells (BMSCs) with lncRNA-Bvht in vitro. Methods: the virus vector p LVX-IRES-Zs Green1-lncRNA-Bvhtcontaining lncRNA-Bvht was constructed to transfect bone marrow mesenchymal stem cells into cardiomyocyte-like cells. LncRNA-Bvht transfection and transfection efficiency were detected. Bone of C57BL / 6 mice was isolated and cultured. Marrow mesenchymal stem cells, The third passage cells were divided into three groups: bone marrow mesenchymal stem cells group, empty carrier group and lncRNA-Bvht group. After 48 h culture, 5-azacytidine was used to induce differentiation for 24 h and then normal culture for 2 weeks. The differentiation of bone marrow mesenchymal stem cells into cardiomyocytes was observed by fluorescence microscope, and the expression of troponin T and ganglion protein, which was the marker of myocardial differentiation, was detected by Western blottqRT-PCR. The expression of troponin T and ganglion protein was strongly positive in lncRNA-Bvht group. The expression of troponin T and ganglion protein in lncRNA-Bvht group was significantly higher than that in blank vector group and bone marrow mesenchymal stem cell group. The results showed that the expression of troponin T and sarcomere protein in lncRNA-Bvht group was significantly higher than that in blank vector group and bone marrow mesenchymal stem cell group. It can effectively promote the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro.
【作者单位】：中山大学孙逸仙纪念医院急诊科;
【基金】：国家自然科学基金(81070125)“抗凋亡与促血管生成miRNA-378干预MSCs治疗心梗后心衰的机制研究”;国家自然科学基金(81270213)“ANGⅡ/AT1/SMAD/CX43通路在心肌干细胞提高心梗大鼠心电生理学稳定性和室颤阈值的作用机制研究”;国家自然科学基金(81670306)“心肌干细胞经IGF-1/STAT/miR-155通路下调AT1R改善心肌梗死后心电生理稳定性机理研究” 广东省科技计划项目(2014A020211002)“LncRNA-Bvht/MESP1/N-cadherin通路调控MSCs向心肌细胞定向分化的机制研究”;广东省科技计划项目(2010B031600032)“抗凋亡与促血管生成miRNA-378干预MSCs治疗心梗后心衰的机制研究” 高校基本科研业务费中山大学青年教师重点培育项目(13ykzd16)“PPAR-γ/TGF-β1/Smad/CX43通路在PPAR-γ干预MSCs治疗心梗后心衰的疗效及机制研究” 广东省医学科研基金(A2016264)”心肌干细胞经由HIF-1α/Apelin/APJ/ACE2通路下调ANGII改善心肌梗死大鼠心电生理学稳定性的机制研究”~~
【分类号】：R54

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